Generation and Analysis of Canine Retinal ESTs: Isolation and Expression of Retina-Specific Gene Transcripts Chung-Tien Lin* ,1 and David R. Sargan† *Department of Veterinary Medicine, National Taiwan University, Taipei, Taiwan; and †Department of Clinical Veterinary Medicine, University of Cambridge, Cambridge, England, United Kingdom Received February 22, 2001 Canine generalized progressive retinal atrophies (gPRA) are a group of degenerative retinal diseases that are a major cause of hereditary blindness in a number of dog breeds. The expressed sequence tag (EST) approach was used to identify and characterize potential candidate genes from canine retinal cDNA libraries. Both conventional and subtractive canine retinal cDNA libraries were constructed and analyzed. Differential hybridization was performed to identify abundantly retinal expressed cDNA clones. Sequences of both random and abundantly expressed clones were analyzed using GCG software and searched against GenEMBL databases. For genes of interest isolated from the libraries, Northern blotting and RT-PCR were performed to determine mRNA expression of the genes. DNA sequences from 85 differentially expressed clones and 100 random cDNAs were obtained and an- alyzed. A higher percentage of abundantly retina- expressed clones showed homology to database se- quences compared with random clones (72 versus 43%). Five retinal genes and 2 anonymous retinal ESTs were selected to analyze mRNA expression. The five known genes, namely HRG4/unc119, cGMP-PDEA, transducin 1A, opsin, and sFRP2 showed retina- specific expression. In anonymous ESTs, clone p81 re- vealed retina-specific expression, while p3 showed ex- pression in each of 14 canine tissues. Transcripts of the canine secreted frizzled related protein 2 (sFRP2) gene showed surprisingly high abundance in the ca- nine retina. The isolated retinal ESTs here will be useful resources for further investigation of canine retinal function and canine genome mapping. © 2001 Academic Press Key Words: retinal degeneration; progressive retinal atrophy; EST; candidate genes; secreted frizzled re- lated protein; dog. Hereditary retinal dystrophies affect a variety of mammals, including humans, dogs, cats, and mice. In the canine model of these diseases, canine progressive retinal atrophies (gPRA or PRA) are a group of hered- itary retinal diseases causing blindness in a number of dog breeds. In human medicine, the equivalent dis- eases are termed Retinitis Pigmentosa (RP), a major group of inherited retinal diseases leading to blindness in humans. Both human and canine retinal degenerations are extremely heterogeneous genetically and clinically. For canine gPRA, a number of retina-specific genes encod- ing proteins involved in visual transduction cascade in photoreceptors are thought to be potential candidates for the disease. Two different mutations in cGMP- PDE gene in the Irish setter and Sloughi breeds (26, 8, 9), a mutation in PDE gene in the Cardigan Welsh corgi (20), a mutation in RPGR in the Siberian Husky (31) and a mutation in RPE65 in the Briard (4, 27) have all been implicated as causing gPRA in a breed specific manner. A number of other retinal genes have been investigated and excluded as causes of gPRA in the dog breeds examined: for example, rhodopsin (11, 28); RDS/peripherin (23); rom-1 (12); cGMP-PDE and cGMP-PDE (1, 29), and transducin- 1 subunit (24). Most gene defects causing gPRA are still unknown. Expressed sequence tag (EST) based candidate gene approach was used in this study to identify and char- acterize potential canine candidate gene homologues known to be the causal genes for human RP. The EST approach is an efficient way of identifying new genes and describing the transcription activity of a tissue (2, 3). Based on findings in other species, retina- specifically expressed genes form one of the most im- portant groups of candidate genes for inherited retinal degeneration. Besides, subtractive hybridization and differential screening of ESTs are useful ways of iden- tifying differentially expressed genes. We are aiming to identify more retina-specific genes and analyze their expression profiles from the canine retinal gene popu- 1 To whom correspondence should be addressed at Department of Veterinary Medicine, National Taiwan University, No. 142, Chou- San Road, Taipei 106, Taiwan. Fax: 886-2-27359931. E-mail: ctlin@ccms.ntu.edu.tw. Biochemical and Biophysical Research Communications 282, 394 – 403 (2001) doi:10.1006/bbrc.2001.4587, available online at http://www.idealibrary.com on 394 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.